In vivo affinity maturation of the HIV-1 Env-binding domain of CD4

Abstract

Many human proteins have been repurposed as biologics for clinical use. These proteins have been engineered with in vitro techniques that improve affinity for their ligands. However, these approaches do not select against properties that impair efficacy such as protease sensitivity or self-reactivity. Here we engineer the B-cell receptor of primary murine B cells to express a human protein biologic without disrupting their ability to affinity mature. Specifically, CD4 domains 1 and 2 (D1D2) of a half-life enhanced-HIV-1 entry inhibitor CD4-Ig (CD4-Ig-v0) were introduced into the heavy-chain loci of murine B cells, which were then adoptively transferred to wild-type mice. After immunization, transferred B cells proliferated, class switched, affinity matured, and efficiently produced D1D2-presenting antibodies. Somatic hypermutations found in the D1D2-encoding region of engrafted B cells improved binding affinity of CD4-Ig-v0 for the HIV-1 envelope glycoprotein (Env) and the neutralization potency of CD4-Ig-v0 by more than ten-fold across a global panel of HIV-1 isolates, without impairing its pharmacokinetic properties. Thus, affinity maturation of non-antibody protein biologics in vivo can guide development of more effective therapeutics.

Fig. 1.. Engineering primary murine B cells to express a B-cell receptor with CD4 domains 1 and 2.

a A representation of an engineered BCR with a potency and half-life enhanced form of CD4 domains 1 and 2 (D1D2) fused through a (G₄S)₃ linker to the amino-terminus of the heavy-chain variable region of the mouse antibody OKT3 (D1D2-OKT3-V_H). The OKT3 heavy chain pairs with an endogenous mouse light chain. b Introducing D1D2-OKT3-V_H at the murine heavy-chain locus. The CRISPR effector protein Mb2Cas12a targets the J4 coding region 5’ of a CTTA PAM, as represented. An rAAV-delivered homology directed repair template (HDRT) complements the 5’ UTR of a VH segment and the intron 3’ of JH4 using 576 bp and 600 bp homology arms, respectively. The edited genome replaces the VDJ-recombined heavy chain with a cassette encoding D1D2-OKT3-V_H. c Expression of D1D2-OKT3-V_H in primary mouse B cells. Expression of D1D2 in edited cells was measured by flow cytometry with monomeric HIV-1 gp120. Representative flow cytometry plots of B cells edited with HDRT targeting the 5’ UTR of VH1–34 or V1–64 were generated 48 h after electroporation. HDRT were delivered with rAAV transduced at 10⁴ multiplicity of infection (MOI). Controls include cells electroporated with Mb2Cas12a ribonucleoproteins (RNP) without rAAV (No HDRT) or without gRNA but transduced with HDRT-encoding rAAV (No gRNA). Plots were gated on viable singlet B cells. d Quantitation of editing efficiency in c from independent experiments. Each dot represents an average from two biologically independent replicates. Error bars represent standard error of mean (SEM). Statistical significance was determined by two-way ANOVA followed by H-Šídák’s multiple comparisons (****p < 0.0001). a, b are created with BioRender.com.

Fig. 2.. Engineered B cells generated neutralizing responses in immunized mice.

a Schedule of immunization and blood collections from mice analyzed in subsequent figures. Naïve B cells from CD45.1 donor mice were engineered ex vivo and 5 million cells per mouse were adoptively transferred to CD45.2 recipient mice 24 h later. Mice were immunized with SOSIP-TM (16055-ConM-8.1) mRNA-LNP on two-week (2 wk, Day 2, 16, and 30, n = 5) or four-week (4 wk, Day 2, 30, 58, n = 5) intervals, and serum was collected seven days after each immunization. Spleens and lymph nodes were harvested four days after the final immunization and B cells isolated from these tissues were analyzed by flow cytometry and next-generation sequencing (NGS). b, c Neutralizing responses of sera from mice immunized with SOSIP-TM mRNA. Sera from mice immunized at two-week (b) or four-week (c) intervals were measured individually for their ability to neutralize BG505 HIV-1 pseudovirus in TZM-bl cell assays. Sera from mice (n = 3) engrafted with unedited CD45.1 B cells and immunized on two-week interval (grey dots) served as negative controls. 100 μg/ml CD4-Ig-v0 combined with normal mouse serum served as a positive control. Dots and error bars indicate median and interquartile range for each group. d A summary of the 50% inhibitory dilutions (ID₅₀) of sera from each immunized mouse in b and c. Statistical significance was determined using repeated-measures two-way ANOVA with Geisser-Greenhouse correction. e Serum concentration of CD4-OKT3-IgG after each immunization, measured by ELISA with an anti-CD4 antibody and CD4-OKT3-IgG as the standard.

Fig. 3.. Engineered B cells persisted in vivo following immunization.

a Quantification of CD45.1-positive donor B cells in vaccinated mice. Four days after the final vaccination of mice characterized in Fig. 2, B cells were isolated from their lymph nodes and spleens. B cells were analyzed by flow cytometry. Figure shows the percent of CD45.1-positive donor cells in mice immunized at two-week (2 wk, n = 4) or four-week intervals (4 wk, n = 5). Mice similarly engrafted with unedited cells and immunized in parallel (Mock, n = 3) served as the controls. For gating strategies, see Extended Data Fig. 3a. b A greater proportion of CD45.1 donor cells binding to HIV-1 gp120. The cells analyzed in panel a were measured for binding to HIV-1 gp120. See Extended Data Fig. 3b for source flow cytometry analysis. c Enrichment of gp120-binding donor cells in the germinal center. Germinal center (GC) B cells (CD38- GL7+) were analyzed by flow cytometry for their ability to bind gp120 and an anti-CD45.1 antibody. Two representative examples (M1 and M2) from mice immunized at two-week interval are shown. Additional examples are provided in Extended Data Fig. 3d. d Quantification of results from experiments shown in c (n = 4). e Distribution of isotypes among D1D2-expressing donor B cells isolated after the final immunization compared with edited donor B cells before engraftment, as determined by NGS. M6 and M7 was combined as one sample. For a, b, d, error bars indicate SEM. Statistical significance was determined by generalized linear mixed model followed by Tukey HSD pairwise comparisons (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig. 4.. D1D2-expressing B cells hypermutated and class switched in vivo.

a Nucleotide mutation frequency across the D1D2-encoding region. mRNA isolated from CD45+ IgG+ gp120-binding B cells from each of 10 mice was analyzed by NGS and the mean frequency of nucleotide mutations is plotted for each position. Triangles represent the most frequent coding mutations. Codons for R59 and K90 are indicated. b Distribution of synonymous (Syn) and non-synonymous (Non-syn) mutation frequency across D1D2. Each dot represents the mutation frequency at one nucleotide position, averaged on five mice in each group. The top two dots indicate the nucleotide mutations leading to R59 and K90 mutations. Line indicates the median. c Average number of accumulated mutations per unique sequence. Significance was determined by two-tailed unpair t test. d The frequency of synonymous mutations within domains 1 (D1) and 2 (D2) for mice immunized at two-week (2 wk) and four-week (4 wk) intervals. The center line indicates mean, and boxes denote quartile range. Repeated measure mixed effects analysis with H-Šídák’s multiple comparisons (*p < 0.05). e Distribution of accumulated nucleotide mutations per unique D1D2 sequence. The center line indicates mean, and boxes denote quartile range. Statistical significance in b, e was determined by mixed effects analysis with H-Šídák’s multiple comparisons (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Fig. 5.. Diverse and convergent amino-acid mutations in engrafted mice.

a The frequency of amino acid changes across D1D2 sequences from each mouse (M1 through M10). Three amino acids with the highest mutation rate are labeled. M6 and M7 were combined in sequencing analysis. b Amino acid changes found in the eight most frequently mutated D1D2 residues from the indicated mice immunized at two-week intervals (M1, M2) are represented. The data for remaining mice are shown in Extended Data Fig. 5. c Minimum spanning trees of D1D2 sequences from individual mice. Each tree presents the inferred lineage and all amino-acid mutations found in M1 and M2. The central black dot represents the inferred ancestral sequence which corresponds to the input sequence. Each circle indicates a distinct amino-acid sequence. Circle size is proportional to the number distinct nucleotide sequences with the same translation. Colored circles mark translations encoded by the eighteen largest number of distinct sequences, with the rank order indicated by number. Branch length corresponds to evolutionary distance, defined as the number of amino-acid differences. The figures for the remaining mice are provided in Extended Data Fig. 6.

Fig. 6.. In vivo hypermutations in D1D2 improve the neutralization potency of CD4-Ig-v0.

a Neutralization potency of CD4-Ig-v0 and its variants modified with R59K, K90R, or combined against the indicated isolates in TZM-bl assays. Curves are shown in Extended Data Figure 7a. Statistical significance was determined by two-way ANOVA with Dunnett’s multiple comparisons. b Location of selected D1D2 mutations (lime) shown on a structure of CD4 (yellow) bound to an HIV-1 Env trimer (light grey). Structure was adapted from pdb:5U1F. c Representative neutralization curves of CD4-Ig, CD4-Ig-v0 and v1-v4 variants bearing combinations of mutations listed below the figure, against a 12-isolate global panel of HIV-1 pseudoviruses. All curves were fitted with a variable slope four parameters dose response model. d IC₅₀ of CD4-Ig, CD4-Ig-v0 and the indicated engineered (v1-v4) and naturally emerging variants (M1–1, M3–1, M6/7–1) against the global panel (****p < 0.0001). Each dot represents an average of two independent experiments. Center lines indicate median. Statistical significance in d was determined by repeated-measures two-way ANOVA with Dunnett’s multiple comparisons.

Fig. 7.. CD4-Ig variants bind Env trimers with higher affinity than CD4-Ig-v0.

a Fitted sensorgrams show one of two replicates of the indicated CD4-Ig variant binding to 16055-ConM-v8.1 SOSIP trimers. Anti-human IgG antibodies were immobilized on the surface to capture CD4-Ig. After CD4-Ig capture, SOSIP protein was injected at concentrations of 800, 400, 200, 100, 50 nM in single-cycle kinetics at 25°C. The experimental data were fitted with a 1:1 Langmuir model. b Summary of K_off, K_on and K_d for CD4-Ig variants. K_d is calculated from K_off and K_on.